Recent data indicate that most, if not all, solid cancers have a hierarchical structure and contain a small number of cancer stem or cancer initiating cells (CICs). Only this population has the ability to self-renew and to repopulate a tumor while their progeny lack this ability. Elimination of all CICs will therefore be required for cancer cure. Cell populations enriched for CICs have been recently prospectively identified in brain tumors, breast cancer, prostate cancer, colon cancer, pancreatic cancer, cancer of the head and neck, and melanoma based on surface marker profiles. However, so far no marker has been found that uniquely distinguishes individual CICs. In our preliminary data, we report the discovery of a fundamental difference between CICs and non-CICs, namely that CICs have little if any 26S proteasome activity. We discovered this using a reporter gene system that targets the ZsGreen protein for degradation specifically through the 26S proteasome by virtue of the addition of a carboxy-terminal degron of ornithine decarboxylase. We have used this system to trace CICs in glioblastoma cell lines and to study their response to cancer treatment in vitro and in vivo. Furthermore, we have used the same degron to destabilize thymidine kinase and to target it using ganciclovir, in so doing eliminating CICs from glioblastoma cell populations. We hypothesize that this fundamental difference in proteasome activity between CICs and non-CICs contributes to the relative resistance of CICs against established therapy modalities and that targeting low 26S proteasome activity can be utilized to treat the most relevant and important subpopulation of cells within a cancer mass. In this project, we will further characterize CICs in glioblastoma with respect to low proteasome activity and how it relates to expression of other stem cell markers. We will investigate the mechanisms that lead to low 26S proteasome function in CICs and whether this is associated with the type of proteasomes that are expressed. We will investigate the functional relationship between low proteasome activity and radiation resistance. Finally, we will explore the underlying mechanisms and we will modify the involved pathways in order to increase proteasome activity and radiation sensitivity. PUBLIC HEALTH RELEVANCE: Cancer stem cells are relatively resistant to conventional anti-cancer therapies and are believed to be the only cells in a tumor responsible for most treatment failures. Currently, tumors have to be explanted and disintegrated to detect cancer stem cells. This proposal uses a novel feature of cancer stem cells to make them visible in living animals, investigates the underlying mechanisms, and applies this feature to understand the resistance of these cells to cancer therapies like radiation.